Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA : colloidal gold by Hep G2 cells

Previously, we have identified a membrane-mediated uptake and translocation of 1,3,5(10)-estratrien-3, 17 beta -diol 6-(O-carboxymethyl)oxime:I-125-labeled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plasma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction). To further investigate this unique effect, current experiments have involved the use of 1,3,5(10)-estratrien-3, 17 beta -diol 17-hemisuccinate: I-125- BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and translocation of the ligand in human hepatoblastoma (Hep G2) cells. In addition, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to directly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affinity, stereospecific binding of E17(125)I-BSA, This same ligand was also translocated from the P3 fraction to the P2 fraction. In contrast,I-125-BSA was minimally removed from the culture medium. Electron micrographs of Hep G2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clathrin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multi-vesicular bodies, suggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min postexposure, the ligand could be viewed in organelles, many of which had vesiculated interiors, resembling rounded, vesiculated mitochondria. labeled BSA was detected mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually eliminated by 100 nM unlabeled E17 BSA or free 17 beta -estradiol, but not 17 alpha -E6 BSA, 17 alpha -estradiol or P6 BSA, and also by exposure of the cells to reduced temperature. These experiments are the first to visually demonstrate membrane binding and specific uptake of an estrogen-containing ligand while allowing the intracellular structures responsible to be seen. Furthermore, they identify a potentially new pathway of receptor-mediated endocytosis; namely, the shuttling of estrogens to the mitochondria, in addition to the classical lysosomal pathway.